Coagulation factor VII (FVII) is a precursor of serine protease that causes blood to clot in the coagulation cascade where it activates coagulation factor X or factor IX. FVII, produced in the liver, is a glycoprotein which consists of a single chain with a molecular weight of 50,000 Da. When decomposed into two chains by different proteases, among which are factor Xa, factor XIIa, factor IXa and thrombin, FVII is itself activated into an active form, FVIIa. FVII is also activated to FVIIa by binding to tissue factors and negatively charged phospholipid A (Nemerson et al, Thromb. Res, 1985, 40:351˜358). In the presence of tissue factor and calcium ions, FVIIa activates factor X into factor Xa which, in turn, convert prothrombin into thrombin with the aid of factor Va in the presence of calcium ion and phospholipid, performing coagulation.
Factor VII, consisting of 406 amino acids, is cleaved at the peptide bond between arginine 152 and isoleucine 153 to form factor VIIa in which the light chain (152 amino acids) and the heavy chain (254 amino acids) are held together by a disulfide bond. The light chain comprises one gamma carboxyl glutamic acid domain (Gla) and two EGF (epidermal growth factor) domains while the heavy chain is responsible for serine protease activity.
In order for Factor VII to be fully functional, it must be subjected to gamma-carboxylation of 10 n-terminal glutamic acid residues, which is a vitamin-K dependent process (Hagen et al, Natl. Acad. SC. U.S.A, 1986, 83:2412˜2416). This Gla-domain is known to be involved in binding factor VII to the cell surface containing the tissue factor (Sakai et al, J. Biol Chem, 1990, 265:1890˜1894).
To date, there are two approaches to the production of factor VIIa. In one approach, factor VII is isolated and purified from plasma and activated to VIIa (Broze et al, J. Biol. Chem, 1980, 225:1242˜1247). The other is a genetic engineering technique in which animal cells transformed with a DNA sequence of factor VII are cultured to produce factor VII (European Patent Application No: 86302855.1).
The plasma-derived factor VIIa suffers from the disadvantage of having a low production yield and lacking consistency in supply. A particular problem with this factor lies in the risk it poses to the safety of the body. In contrast, the genetic recombinant product is considered to overcome the demerits of the plasma-derived product.
However, animal cells from which factor VII is produced by genetic recombination generally have a low expression level, so that only low productivity can be guaranteed. Hence, in order for factor VII to be used as a therapeutic agent, it is necessary to secure a cell line in which the factor can be stably produced on mass scale. In this context, an expression vector that has high expression efficiency is indispensable.